The field of this invention is recombinant procollagens containing novel cleavage sites.
Collagen has provided a number of commercial applications, including use as a convenient substrate for cell culture, as well as in the manufacture of biocompatible materials (e.g., artificial skin) having a variety of therapeutic applications in humans. Commercially available collagens are primarily isolated from freshly killed farm animals (e.g., cows, pigs, chickens) in polymerized form or in soluble form derived by enzymatic or chemical treatment of polymerized collagen. However, polymerized collagens, and soluble collagens derived from polymerized collagens, are of limited usefulness due to the presence of natural crosslinks within the collagen. Furthermore, non-human collagens can provoke undesirable immune responses when administered to human subjects.
As one alternative, human collagen can be purified from human sources such as human placenta, as described in U.S. Pat. No. 5,002,071 (Research Development Foundation) and in copending U.S. application Ser. No. 07/921,810 (Collagen Corporation). In addition to source limitations and the risk of contamination by human pathogens such as Hepatitis viruses and HIV, the methods for recovering collagen from these sources bias the type of collagen recovered. Furthermore, because the collagen is derived from naturally crosslinked tissues, the collagen recovered is not entirely homogeneous. Another approach to the production of human collagen is the expression of recombinant human collagen in the milk of transgenic animals, as described in copending U.S. applications Ser. Nos. 08/183,648 and 08/011,643 (Collagen Corporation). However, this approach subjects the recombinant collagen to any host deficiencies in translational processing.
The term collagen refers to a type of protein that encompasses a class of structurally related polypeptides consisting of helical collagen chains and homo- and heteromeric polymers thereof. Collagen chains often contain both long helical domains and non-helical extensions, or telopeptides. The helical domains comprise -(GXY)n- repeats, where X and/or Y are frequently proline or hydroxyproline.
Collagen polypeptide chains are encoded by a number of related genes. See S. L. Adams, Amer. J. Respir. Cell and Mol. Biol. (1989) 1:161-168; Mayne and Brewton, Current Opinion in Cell Biology (1993) 5:883-890; and van der Rest and Bruckner, Current Opinion in Structural Biology (1993) 3:430-436. Collagen-encoding transcripts are initially translated into procollagen chains which undergo a variety of post-translational events such as processing, secretion, and assembly (e.g., disulfide exchange, prolyl residue hydroxylation, glycosylation, and crosslinking of the helical chains; see, for example, Prockop et al., New England J. Med. (1984) 3111:376-386) to form mature collagen chain helices. In addition, procollagens contain N- and C-terminal propeptides that facilitate collagen formation. See, for example, Lee et al., J. Biol. Chem. (1992) 267: 24126-24133.
Many of these post-translational events are catalyzed by enzymes thought to be expressed only in or by cells which naturally produce collagen. For example, the procollagen propeptides are cleaved from the procollagens by tissue-specific procollagen N- and C-terminal proteases. Accordingly, recombinant production of collagen has been severely restricted by the inability of conventional cellular expression systems to efficiently post-translationally modify procollagen to form functionally mature collagen.
Site-specific proteolysis of unrelated fusion proteins is reported by Paul Carter in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, American Chemical Society Research Symposium (1990) 47:181-193; Brenner and Fuller, PNAS (1992) 89:922-926; Seeboth et al., Appl. Microbiol. Biotechnol. (1992) 37:621-625; and Liu and Miller, WO 8912678. Chemical cleavage is described in Chapter 2 of Fontana and Gross, In Practical Protein Chemistryxe2x80x94A Handbook, Darby, Ed., John Wiley and Sons, London (1986).
The invention provides recombinant procollagen chains having a natural collagen polypeptide chain separated from a propeptide by a non-natural site-specific proteolytic agent recognition site (i.e., the site is not naturally present at that location of a natural procollagen chain). The propeptide is often a C-terminal propeptide and, frequently, a natural procollagen C-terminal propeptide. Alternatively, the recombinant procollagen chains may further comprise a second propeptide separated from the opposite end of the natural collagen chain by a second non-natural site-specific proteolytic agent recognition site.
A wide variety of propeptides and site-specific proteolytic agent recognition sites may be used. The propeptide is capable of increasing the yield of at least one of secreted procollagen (trimeric procollagen chains) or collagen monomers (trimeric collagen chains) from cells expressing procollagen chains comprising the propeptide, as compared with cells expressing the same collagen chains alone, without the propeptide. The non-natural site-specific proteolytic agent recognition site is generally selected to provide a substrate for a convenient site-specific proteolytic agent. The agent is commonly a protease or a chemical agent which selectively cleaves at accessible and preferentially labile peptide bonds. Where two site-specific proteolytic agent recognition sites and two propeptides are present, the recognition sites may have the same or different amino acid sequences, with the same or different site-specific proteolytic agent affinities and specificities.
The subject procollagen chains may be constructed such that the site-specific proteolytic agent cleavage site is a peptide bond that separates the collagen chain and/or the propeptide from the remainder of the procollagen chain. Alternatively, the cleavage site may be a peptide bond that separates, from the remainder of the procollagen chain, the collagen chain (or propeptide), joined to at least one amino acid which is not naturally present at that location of a natural collagen chain (or propeptide).
The invention provides collagens produced by contacting the subject recombinant procollagen with site-specific proteolytic agents capable of selectively cleaving the procollagen chains at the site-specific proteolytic agent recognition sites, wherein the resulting chains comprise terminal peptides which may have non-natural amino acid sequences (i.e., not naturally present at that location of a natural collagen chain). The resulting collagen may be employed directly (e.g. to coat cell culture dishes or cultureware) or further polymerized and/or provided in sterile, nontoxic, biocompatible collagen compositions suitable for administration to humans.
The invention also provides nucleic acids encoding the subject procollagen chains, as well as vectors and cells comprising such nucleic acids operably linked to transcription regulatory elements not naturally linked to such nucleic acids. The invention provides processes for the production of recombinant procollagen and/or collagen chains in and from such cells. The invention also provides methods of utilizing the resulting procollagen and collagen, for example, coating cultureware and augmenting localized tissue in a host by, for example, subcutaneous administration.